Cloning and characterization of Trimeresurus gracilis venom phospholipases A ₂: Comparison with Ovophis okinavensis venom and the systematic implications

This study focuses on the structural and functional characterizations of novel venom phospholipases A ₂ (PLA ₂s) from Trimeresurus gracilis, an endemic Taiwanese pitviper. The PLA ₂ cDNAs were cloned from venom glands and sequenced. The majority of the clones encoded a Glu6-containing PLA ₂ (designated as Tgc-E6) whose deduced amino acid sequence resembled those of other Crotalinae acidic PLA ₂s. Tgc-E6 was also purified and constituted about 6% (w/w) of the total venom proteins. For human platelet rich plasma, Tgc-E6 inhibited the ADP- and collagen-induced aggregation with an IC ₅₀ of 272nM and 518nM, respectively. Like Ovophis okinavensis venom, T. gracilis venom did not contain any Lys49-PLA ₂s, although a cDNA encoding Lys49-PLA ₂ has been cloned from each of the species. Their predicted protein sequences are 94% identical, and their pI values 8.3 are lower than those of other Lys49-PLA ₂s, mainly due to the acidic substitutions within positions 78-111, which are apparently more similar to those in Tgc-E6 than to those in other Lys49-PLA ₂s. This unique structural feature of the venom PLA ₂s thus render evidence for close phylogenetic relationship between both species. The structural variations in the venom acidic PLA ₂s of the two species possibly have resulted from adaptation to different prey ecology.